Abstract: Metastasis remains the major cause of cancer mortality, but breakthroughs in our understanding of the molecular and cellular mechanisms regulating metastasis have yet to be broadly translated into improved survival rates in patients with metastatic disease. The challenge is how to treat cancer cells that have spread to lymph nodes or distant organs in order to prevent their growth and ideally eradicate them from the body. Most cancer therapies are developed against the primary tumor growing in its native microenvironment. However, it is clear that the local microenvironment in which tumor cells grow greatly affects the growth rate, metabolism, vascularization and ultimately response to therapeutic intervention. In this NIH Director's New Innovator Award, I propose to biologically characterize the growth of metastatic tumor cells in lymph nodes starting from individual seeded cancer cells. This research is motivated by basic, critically important questions that will ultimately help clinicians better manage metastatic disease: i) What are the biological triggers for metastatic growth in lymph nodes? ii) Are lymph node metastases clonal or are multiple cancer cells needed to initiate growth in the node? iii) Is there a therapeutic benefit to the removal lymph nodes with only a few cancer cells? iv) Do lymph node metastases seed distant sites leading to further dissemination? To address these questions, I will utilize our newly developed chronic lymph node window in order to study spontaneously disseminated cancer cells in the lymph node. This innovative window model overcomes a major barrier to research and allows us to monitor the earliest stages of metastatic growth in lymph nodes, characterize the biological triggers for growth from individually seeded cancer cells, determine whether lymphatic metastasis are clonal and investigate whether lymph node metastases can further disseminate. In this way, we will use novel techniques to address the problem of lymphatic metastasis in a new way-characterizing a growing metastasis in its new microenvironment. These clinically driven projects will yield data to help i) better predict outcome and design therapeutic courses for patients with micrometastatic disease in their lymph nodes, as well as ii) uncover the underlying biology of metastatic tumor growth in the lymph nodes, potentially leading to novel targets for the treatment of lymphatic metastasis. Based on my foundational background in engineering, along with over 12 years experience innovating intravital microscopy techniques and animal models to study critical problems in tumor metastasis, I am uniquely qualified to lead this effort. I have used these skills to creatively solve problems that have hindered research in critical areas of lymphatic biology and lymphatic metastasis. I have shown that I am willing to challenge conventional paradigms and keep my work focused on clinically relevant problems that need basic biological discoveries to drive new therapies. I have also shown I can work with clinicians to impact patient care. I will continue to build on my past successes with this proposed NIH Director's New Innovator Award and hopefully have a positive impact on the treatment of patients with lymphatic metastases. Public Health Relevance: Metastasis remains the major cause of cancer mortality posing the challenge of how to treat cancer cells that have spread to lymph nodes or distant organs in order to prevent their growth and ideally eradicate them from the body. To answer this challenge, I will utilize our newly developed chronic lymph node window to characterize the triggers for the growth of seeded cancer cells and investigate whether lymph node metastasis can further disseminate. These clinically driven projects will yield data to i) better predict outcome and design therapeutic courses for patients with lymphatic metastases, as well as ii) uncover the underlying biology of metastatic tumor growth in the lymph nodes, leading to novel targets for the treatment of lymphatic metastasis.